CN103031279B - Recombinant flavivirus vaccines - Google Patents

Abstract

The invention provides the recombinant flavivirus vaccines that can be used for prevention and therapy flaviviridae infections.Vaccine of the present invention contains the recombinant flavivirus comprising Attenuating mutations.

Description

Recombinant flavivirus vaccines

The divisional application that the application is the applying date is on 04 24th, 2006, Chinese application number is 200680022652.9, denomination of invention is the patent application of " recombinant flavivirus vaccines ".

Background of invention

The present invention relates to the vaccine containing recombinant flavivirus.

Flavivirus is tunicary positive chain RNA small virus, and it is propagated by the mosquito be infected and tick usually.Some flavivirus, such as yellow fever virus, dengue fever virus, japanese encephalitis virus, Tick-borne encephalitis virus and west Nile virus, threatening to the public health in the whole world or may threaten.Such as, yellow fever virus has become the epiphytotics cause of disease in some jungle region in Sub-Sahara Africa and some areas of South America.Although it is gentle that many yellow fever viruses infect, this disease also can cause serious, life-threatening disease.Initial or the acute phase of this state of an illness, is usually expressed as high heat, shiver with cold, headache, backache, myalgia, appetite stimulator, nausea and vomiting.After 3 to 4 days, these transference cures.In some patient, symptom occurs again afterwards, because disease enters its so-called toxicity phase.In this stage, again there is high heat and shock, hemorrhage (such as from mouth, nose, eye and/or gastrorrhagia), renal failure and liver failure can be caused.Even, liver failure causes jaundice, and namely yellowing of the skin and eyes turn white, gain the name thus " yellow jack ".The nearly half of patient entering the toxicity phase is dead in 10 to 14 days.But, from the people of yellow jack recovery from illness, there is the immunizing power of resisting again subinfection all the life.The number infected by yellow fever virus in the past twenty years rises to some extent, has about 200 every year so far, 000 routine yellow fever cases, about 30,000 routine associated death.Therefore, yellow fever virus again there are the serious problems becoming publilc health.

West Nile (WN) virus is distributed widely in Africa, the Indian subcontinent, Europe, Ukraine, Russia, the Central Asia and the Middle East (Monath and Heinz, in the 3rd edition Virology that the people such as Fields compile, Lippincott-Raven, pp.961-1034,1995).1999, there occurs once the beyond example epidemic encephalitis (Enserik, Science286:1450-1451,1999) by the WN people that causes of virus and Ma in the U.S..From that time, this virus has forever captured the U.S., has attacked almost whole territories of the U.S..The record of sickness rate so far/mortality ratio aspect results from 2003, reports 9862 routine cases, and wherein about 1/3rd with neurological symptoms result, also has 264 examples dead.The disease of people changes from the singapore hemorrhagic fever sample disease of gentleness to fatal meningoencephalitis, and the most serious disease occurs in the elderly.Up to now, not for the active drug methods for the treatment of of west Nile virus, and to monitor and prevention method can not the case load of remarkably influenced human infection.Virus transport enters South America and high in the risk that under-developed country is popular.Develop vaccine safely and effectively and control the popular of future by contributing to.

Flavivirus, comprises yellow fever virus and west Nile virus, has two main biological characteristicses to cause it in humans and animals, bring out symptom.First in these two characteristics is close neuro (neurotropism), and namely virus tends to the nervous tissue of invasion and attack and infection host.Neurotropism flaviviridae infections can cause the inflammation of brain and spinal cord and damage (i.e. encephalitis), consciousness to go down, benumb and faint from fear.Second in these biological characteristicses of flavivirus is viscerotropism tropism (viscerotropism), and namely virus is tended to invasion and attack and infected important internal organs, comprises liver, kidney and heart.Viscerotropism flaviviridae infections can cause inflammation and the damage of liver (hepatitis), kidney (ephritis) and cardiac muscle (myocarditis), causes exhaustion or the dysfunction of these organs.

Parent neuro and viscerotropism tropism seemingly flavivirus uniqueness with difference characteristic.Some flavivirus is neurophilic (such as west Nile virus) mainly, and some is viscerotropic (such as yellow fever virus and dengue fever virus) mainly, also has some two kinds of characteristics all to show (such as Kyasanur Forest Disease).But, close neuro and viscerotropism tropism have existence to a certain degree in all flaviviruss.In host, likely there is the interaction between viscerotropism tropism and close neuro, because infect before internal organ occur in and invade central nervous system.Therefore, close neuro depends on the ability that virus copies in extraneural organs's (internal organ).Copy outside this nerve and cause viremia, then cause and invade brain and spinal cord.

The ripe virion of flavivirus through processing generation completely comprises three kinds of structural protein, capsid (C), film (M) and coating (E).7 kinds of Nonstructural Proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) are produced in the cell be infected.Virus receptor combines and is all present in E protein both Fusion domain.In addition, E protein or the ideal composition of flavirirus vaccines, because can neutralize the infectivity of virus for the antibody of this protein and give host the provide protection of resisting this disease.The immature flavivirus virion found in the cell be infected comprises cephacoria (pre-membrane, prM) albumen, and it is the precursor of M albumen.Flavivirus protein produces as follows: translated by single long open reading-frame (ORF) and produce polyprotein; subsequently through the posttranslational protein hydrolysis cutting of polyprotein series of complex; produce ripe virus protein (Ambergetal., J.Virol.73:8083-8094,1999; Rice, " Flaviviridae ", in the Virology that the people such as Fields compile, Raven-Lippincott, NewYork, VolumeI, p.937,1995).Virus structural protein is with the N end regions of the sequential arrangement of C-prM-E at polyprotein, and Nonstructural Protein is then positioned at C end regions with above-mentioned order.

Living vaccine gives for the strongest of the disease caused by virus infection and lasting protective immune response.With regard to flavivirus, the exploitation of successful vaccine needs to change virulence properties, and vaccine virus is weakened the close neuro of human or animal and viscerotropism tropism.Some diverse ways are for the exploitation of the vaccine for flavivirus.With regard to yellow fever virus, have developed two kinds of vaccines (yellow jack 17D and French neurotropism vaccine) (Monath by continuous passage, " YellowFever ", in the 3rd edition Vaccines of Plotkin and Orenstein, Saunders, Philadelphia, pp.815-879,1999).Yellow jack 17D Vaccine is developed by continuous passage in chicken embryo tissue, creates the virus that close neuro and viscerotropism tropism significantly weaken.France's neurotropism vaccine is developed by continuous passage in mouth cerebral tissue, causes losing viscerotropism tropism but retains close neuro.In fact, the high incidence of neuroscience aspect mishap (postvaccinal encephalitis) is relevant with the use of French vaccine.

Another approach of attenuation relates to the structure of chimeric flavirirus, and described chimeric flavirirus comprises the component of two kinds of (or more plant) different flaviviruss.Chimeric flavirirus is prepared into the structural protein and Nonstructural Protein that comprise from different flavivirus.Such as, so-called ChimeriVax ^tMtechnology employs the capsid protein of yellow jack 17D virus and Nonstructural Protein to transport the envelope protein (prM and E) (consulting such as Chambersetal., J.Virol.73:3095-3101,1999) of other flavivirus.In fact; this technology (consults such as Pugachevetal. for exploitation for the candidate vaccine strain that dengue fever virus, Japanese encephalitis (JE) virus, west Nile virus and St. Louis encephalitis (SLE) are viral; in the 3rd edition NewGenerationVaccines that the people such as Levine compile; MarcelDekker; NewYork; Basel, pp.559-571,2004; Chambersetal., J.Virol.73:3095-3101,1999; Guirakhooetal., Virology257:363-372,1999; Monathetal., Vaccine17:1869-1882,1999; Guirakhooetal., J.Virol.74:5477-5485,2000; Arroyoetal., TrendsMol.Med.7:350-354,2001; Guirakhooetal., J.Virol.78:4761-4775,2004; Guirakhooetal., J.Virol.78:9998-10008,2004; Monathetal., J.Infect.Dis.188:1213-1230,2003; Arroyoetal., J.Virol.78:12497-12507,2004; And Pugachevetal., Am.J.Trop.Med.Hyg.71:639-645,2004).These are the virus vacciness of living, and they are similar to YF17D vaccine, cause directly for the strong humoral and cellular immune response response of expection heterologus virus.Based on to ChimeriVax ^tMthe extensive sign of-JE and dengue vaccine, observes ChimeriVax ^tMthe principal character of vaccine is included in substrate cell (substratecell) to copy and reaches high titre (7 ¹⁰pfu/ml or higher) ability; neurovirulence in weanling and minor mouse is low (significantly lower than YF17D); camber attenuation is tested the regular monkey of related neurological virulence and viscerotropism tropism; heredity in vitro and in vivo and phenotypic stability high; duplicating efficiency low (this is extremely important for preventing in the uncontrolled diffusion of occurring in nature) in mosquito, and after using single dose, in mouse, monkey and people, bring out powerful protective immunity and side effect after not having serious immunity.

In other attenuation approach, carry out the mutagenesis of flavivirus (comprising chimeric flavirirus).Some are for making the experimental technique of wild-type flavivirus pathogenic agent attenuation (consult and such as summarize in Pugachevetal., Int.J.Parasitol.33:567-582,2003) on the books.Such as, found to comprise the capsid protein of yellow fever virus and non-structural protein bletilla japanese encephalitis virus, some amino acid whose sudden change of envelope protein of dengue fever virus or the membranin of west Nile virus and the chimeric flavirirus of envelope protein reduced viscerotropism tropism (consulting such as WO03/103571 and WO2004/045529).The large section that another approach being applied to wild-type 4 type dengue fever virus at first to relate in 3 ' non-translational region 30 or more Nucleotide deletes (3'UTR; Menetal., J.Virol.70:3930-3937,1996; U.S. Patent No. 6,184,024B1).These one of are deleted; called after deletes delta 30, d30 or Δ 30; further research (Durbinetal., AJTMH65:405-413,2001 are obtained in the background of wild-type 4 type singapore hemorrhagic fever and 1 type dengue fever virus and 4 types singapore hemorrhagic fever/WN embedded virus; Whiteheadetal., J.Virol.77:1653-1657,2003; Pletnevetal., Virology314:190-195,2003; WO03/059384; WO03/092592; WO02/095075).In addition; find that in wild-type tick-borne encephalitis (TBE) and langat virus (Langatvirus), introduce some large section of 3'UTR deletes (417-616 Nucleotide is long) at mouse model camber attenuation (Mandletal.; J.Virol.72:2132-2140,1998; Pletnev, Virology282:288-300,2001).About the amount of delivering of the vitro data of YF17D vaccine virus is limited.Specifically, the large section that Bredenbeek and coauthor thereof demonstrate all three tumor-necrosis factor glycoproteins (RS) elements of 3 ' UTR deletes that (length is 188 Nucleotide; The position of RS element is as shown in Figure 1A) or the deletion of 25 Nucleotide of conserved sequence elements 2 (CS2) virus can not be stoped to copy in bhk cell, and other deletion (length is 25-68 Nucleotide) of three places affecting CS1 or 3' pole far-end trunk and ring is fatal (Bredenbeeketal., J.Gen.Virol.84:1261-1268,2003).Other data shows to be introduced sudden change and result in attenuation in the 3 ' end trunk-ring macrostructure of flavivirus (singapore hemorrhagic fever) 3 ' UTR, maintain the ability (Markoffetal. of virus immunity host simultaneously, J.Virol.76:3318-3328,2002).

The second method recorded about the attenuation of highly pathogenic wild-type TBE virus make use of deletion relatively large in capsid protein C, described in Kofler and colleague thereof, they introduce a series of deletion and have reclaimed some mutant (Kofleretal. of surviving in the C protein of TBE virus, J.Virol.76:3534-3543,2002).Specifically, 16 amino acid whose deletion (spiral I of prediction in described protein central hydrophobic structural domain; See Fig. 2 A) sharply decrease virus copying and significantly reducing the neural invasiveness in mouse in bhk cell.Highly pathogenic TBE strain Hypr(>100LD is avoided with the immunisation protects mice that this TBE mutant carries out ₅₀) attack (Kofleretal., J.Virol.76:3534-3543,2002).

The vaccine that many medically important flaviviruss with viscerotropism characteristic are not also given the ratification at present, such as west Nile virus, dengue fever virus and msk haemorrhagia fever virus etc.

Summary of the invention

As described herein, the invention provides the recombinant flavivirus (such as yellow fever virus or chimeric flavirirus) comprising a place or many places sudden change, described sudden change makes the viscerotropism tropism of the flavivirus of attenuation have a little reduction.The example of the chimeric flavirirus that the present invention comprises has the capsid protein that comprises the first flavivirus (such as yellow fever virus, such as yellow fever virus strain 17D) and non-structural protein bletilla the second flavivirus (to be such as selected from japanese encephalitis virus, 1 type dengue fever virus, 2 type dengue fever viruss, 3 type dengue fever viruss, 4 type dengue fever viruss, Murray valley encephalitis virus (MurrayValleyencephalitisvirus), Saint Louis' encephalitis virus, west Nile virus, KUN (Kunjinvirus), rocio encephalitis virus (Rocioencephalitisvirus), ILH (Ilheusvirus), CEEV, Siberian encephalitis, RSSE virus, Kyasanur Forest Disease (KyasanurForestDiseasevirus), Alkhurma virus (Alkhurmavirus), msk haemorrhagia fever virus (OmskHemorrhagicfevervirus), (sheep) louping-ill virus (Loupingillvirus), powassan virus (Powassanvirus), negishi virus (Negishivirus), ABS (Absettarovvirus), Hansalova virus (Hansalovavirus), A Bo virus (Apoivirus) and Hypr virus (Hyprvirus)) membranin and the chimeric flavirirus of envelope protein.With regard to comprising the chimeric flavirirus of west Nile virus membranin and envelope protein, envelope protein optionally can comprise replacement at the 107th, 316 and 440 coating amino acid place.The sudden change of recombinant flavivirus of the present invention can be that such as a place or many places delete or replace.

Sudden change of the present invention can be positioned at the region of recombinant flavivirus, comprises 3 ' non-translational region of such as recombinant flavivirus, and usually comprises and be less than 30 Nucleotide.As the example of this type of sudden change, described sudden change can be the sudden change of the possible selectable unit instability making the trunk structure in virus 3 ' non-translational region (trunk structure in the non-conservative district of such as virus 3 ' non-translational region) or the secondary structure predicted or total.As described herein, as concrete example, sudden change can be any one or multiple of d7, dA, dB, dC and dD.In other example, described sudden change can comprise one or more Nucleotide of conserved sequence 2 (CS2), therefore can be such as CS2d5 or CS2d16.In other example, saltant type flavivirus is through revising applicable cell culture substrate (cellculturesubstrate), the spontaneous modification of the sudden change causing it to comprise (is such as deleted, such as such as the deletion of 5 Nucleotide initial in dC mutant is increased to the modification of 24 Nucleotide; See below) or cause the sudden change in second site, described sudden change does not affect attenuation in body.

Sudden change of the present invention also can be introduced in capsid sequence.Such sudden change can comprise 1-3 amino acid whose deletion of such as capsid protein.As an object lesson of this type of sudden change, this sudden change can be that (such as suddenly change C2, as described herein) is deleted in a place in capsid protein spiral I or many places.

In other example, sudden change of the present invention can comprise a place or the many places replacement of coating amino acid.In one example in which, the sudden change of this type of coating is one or more replacement in the 138th, 176,177,244,264 and 280 coating amino acid or its combination.The object lesson of this type of combination comprises the 176th, 177 and 280; 176th, 177,244,264 and 280; With the 138th, 176,177 and 280.

Recombinant flavivirus of the present invention can comprise the sudden change only in one of aforementioned region, or the sudden change in that region two or all three.In addition, recombinant flavivirus can comprise a place or many places sudden change in flavivirus envelope albumen hinge area, and/or comprises sudden change in flavivirus membranin.As the object lesson of recombinant flavivirus of the present invention, be referred to and comprise C2 and suddenly change and combined d7, dB, dD and/or E#5(and E176, E177 and E280) flavivirus of sudden change (seeing below), optionally at ChimeriVax ^tM(also consult hereafter) in the background of-WN02.Other example of the recombinant flavivirus comprising numerous sudden change is provided herein in other place.

Present invention also offers the method for prevention or treatment flaviviridae infections in experimenter (such as people or livestock experimenter), comprise and vaccine containing one or more recombinant flavivirus described herein is used to experimenter, and the medicinal compositions containing this type of recombinant flavivirus.In addition, the present invention includes the nucleic acid molecule (such as RNA or DNA molecular) of the genome (or its complement) comprising this type of recombinant flavivirus.In addition, the present invention includes recombinant flavivirus described herein and prepare the purposes in inactivated vaccine.As described herein, the present invention also comprises the method making flavirirus vaccines Candidate Strain attenuation, is included in the place or many places sudden change that introduce the viscerotropism tropism reducing flavirirus vaccines Candidate Strain in flavirirus vaccines Candidate Strain.

The present invention has some advantages.Virus for the present invention's sudden change is attenuated flavivirus that is alive, that keep the ability of mammalian cell-infecting.Because described virus is infectious, so ensure that their abundant attenuations are important, to make it that the experimenter inoculated can not be caused sick.The invention provides the method for finely tuning candidate's flavirirus vaccines attenuation, making it possible to the vaccine producing safety thus.Recombinant flavivirus of the present invention also has superiority, because they compare safer with parent with wild type strains.This feature for them as the using and using of attenuated vaccine of living, and their preparation and be favourable as the purposes of inactivated vaccine.

According to hereafter describing in detail, accompanying drawing and claims, other features and advantages of the present invention are apparent.

Accompanying drawing is sketched

Figure 1A is the schematic diagram of yellow fever virus 3 ' non-translational region, show the structural domain (tumor-necrosis factor glycoproteins (RS), CS2, CS1 and 3 ' pole far-end trunk-ring structure) in this region, and the example (such as dA, dB, dC, dD, d7, d14, CS2d5 and CS2d16) of the present invention's sudden change.

Figure 1B be yellow fever virus 3 ' non-translational region in the middle part of the 3rd RS element to the sequence of UTR end and the schematic diagram (Proutskietal., J.Gen.Virol.78:1543-1549,1999) of secondary structure.

Fig. 1 C is the schematic diagram of the best YF17D3'UTR secondary structure prediction obtained with ZukerRNA folding algorithm.

Fig. 1 D is that 3'UTR deletes (display dC deletion; Zuker method) schematic diagram on the impact (comparing with Fig. 1 C) of best YF17D structure.

Fig. 1 E is that the size of deleting in dC virus is increased to the schematic diagram of 24 Nucleotide (in P5 level) on the impact (comparing with Fig. 1 D and Fig. 1 C) of the secondary structure of prediction from 5 Nucleotide (in P2 level) are spontaneous.The best YF17D structure that the increase of size of deleting causes similar initial.

Fig. 2 A is Tick-borne encephalitis virus capsid protein sequence and Kofleretal., J.Virol.76:3534-3543, the schematic diagram deleted in this protein of 2002 reports.

Fig. 2 B is the schematic diagram of YF17D capsid protein sequence, indicates region (α spiral I-IV) and the hydrophobic region by Computer Analysis prediction with α helix secondary structure.

Fig. 3 is the schematic diagram of the Homology model of YFE albumen homodimer, shows the position of different residue between wild-type YF (Asibi) and vaccine strain YF17D.

Fig. 4 is the schematic diagram of west Nile virus film (M) albumen and coating (E) albumen, shows the different mutation combination introducing these regions by double-mass model method.

Fig. 5 is the ChimeriVax that display is selected ^tMthe graphic representation of the growth kinetics of-WN04 variant and the large plaque of WN02 (attenuation deficiency vaccine) and the contrast of little plaque.

Detailed Description Of The Invention

The invention provides the recombinant flavivirus that can be used for methods for the treatment of such as method of vaccination.Attenuating mutations is there is in the focusing on of flavivirus of the present invention in viral genome.These sudden changes can make virus attenuation, by viscerotropism tropism and/or the close neuro of such as attenuated virus.Described sudden change can be present in flavivirus genome the region comprising 3 ' non-translational region (3 ' UTR), capsid sequence and/or envelope sequence.Just as further discussed below, sudden change of the present invention can be used for finely tuning the attenuation of the vaccine strain having comprised a place or other Attenuating mutations of many places.Therefore, such as, sudden change of the present invention is by causing in plaque assay plaque size to reduce and/or in animal model, viremia reduces and identified (seeing below).Therefore, sudden change of the present invention provides higher levels of security with regard to the attenuation of this viroid.The details of virus of the present invention and method provide as follows.

An example that can carry out the flavivirus of sudden change of the present invention is yellow fever virus, such as YF17D vaccine strain (Smithburnetal., " YellowFeverVaccination ", WorldHealthOrg., p.238,1956; Freestone, inPlotkinetal. (eds.), Vaccines, 2ndedition, W.B.Saunders, Philadelphia, 1995).Other yellow fever virus strain such as YF17DD (GenBank numbering U17066) and YF17D-213 (GenBank numbering U17067) (dosSantosetal.; VirusRes.35:35-41; 1995), YF17D-204France (X15067; X15062), YF17D-204; 234US (Riceetal.; Science229:726-733,1985; Riceetal., NewBiologist1:285-296,1989; C03700, K02749) and by Galleretal., Vaccine16 (9/10): 1024-1028, the 1998 yellow fever virus strains recorded also can be used for the present invention.

Other flavivirus that can carry out sudden change of the present invention comprises other mosquito matchmaker flavivirus, such as japanese encephalitis virus (such as SA14-14-2), dengue fever virus (serotype 1-4), Murray valley encephalitis virus, Saint Louis' encephalitis virus, west Nile virus, KUN, rocio encephalitis virus and ILH; Tick matchmaker flavivirus, such as CEEV, Siberian encephalitis, RSSE virus, Kyasanur Forest Disease, Alkhurma virus, msk haemorrhagia fever virus, louping-ill virus, powassan virus, negishi virus, ABS, Hansalova virus, A Bo is viral and Hypr is viral; And the virus (such as hepatitis C virus) that hepatitis virus belongs to.All these viruses all have the tendency that some infect internal organs.The viscerotropism tropism of these viruses not necessarily may cause the dysfunction of epochmaking internal organs, but virus can cause viremia these intraorganic copying and cause invasion central nervous system thus.Thus, outside the risk reducing damage internal organs, the viscerotropism tropism being alleviated these viruses by mutagenesis can be reduced their invasion brains and cause the ability of encephalitis.

Except above-mentioned virus and other flavivirus, the chimeric flavirirus comprising a place or many places said mutation type is also included within the present invention.These mosaics can be made up of following flavivirus, one (or more) structural protein the second virus (virus namely to be tested or predetermined, such as flavivirus namely in the first flavivirus (i.e. trunk flavivirus); Consult such as U.S. Patent No. 6,696,281; U.S. Patent No. 6,184,024; U.S. Patent No. 6,676,936; With U.S. Patent No. 6,497,884) the corresponding structural protein of one (or more) replace.Such as, described mosaic can be made up of following flavivirus, film and the coating of the membranin namely in trunk flavivirus (such as yellow fever virus) and envelope protein the second virus to be measured (such as west Nile virus, dengue fever virus (serotype 1,2,3 or 4), japanese encephalitis virus or other virus, all any one virus as referred to herein) replace.Embedded virus can be built by any virus combination, but the virus finding immunity for it is under normal circumstances the source of the structural protein inserted.

The object lesson that one class can carry out the embedded virus of sudden change of the present invention has the Yellow Fever Vaccine strain YF17D of people, membranin wherein and envelope protein are with membranin and the envelope protein replacement of another kind of flavivirus, such as west Nile virus, dengue fever virus (serotype 1,2,3 or 4), japanese encephalitis virus, Saint Louis' encephalitis virus, Murray valley encephalitis virus or other flavivirus any, one of virus such as enumerated above.The chimeric flavirirus built in this approach is called after so-called " ChimeriVax " virus.Use ChimeriVax ^tMtechnique construction is also preserved in American type culture collection (AmericanTypeCultureCollection according to the clause of budapest treaty (BudapestTreaty); ATCC; Manassas; Virginia, U.S.A.), grant the following chimeric flavirirus in January 6 1998 preservation day and can be used for preparing virus of the present invention: yellow fever virus 17D/2 type dengue fever virus embedded virus (YF/DEN-2; ATCC numbering ATCCVR-2593) and yellow fever virus 17D/ japanese encephalitis virus SA14-14-2 embedded virus (YF/JEA1.3; ATCC numbering ATCCVR-2594).Prepare the details of embedded virus used in the present invention see such as following publication: WO98/37911; WO01/39802; Chambersetal., J.Virol.73:3095-3101,1999; WO03/103571; WO2004/045529; U.S. Patent No. 6,696,281; U.S. Patent No. 6,184,024; U.S. Patent No. 6,676,936; With U.S. Patent No. 6,497,884.

As mentioned above, new mutant of the present invention is present in flavivirus and comprises in 3 ' UTR of chimeric flavirirus, capsid and/or envelope sequence.These types sudden change in each, mutually can combine and/or be combined with other Attenuating mutations, be described below:

3 ' non-translational region sudden change

The structure of the 3 ' UTR of yellow fever virus vaccine strain YF17D as shown in Figure 1A, is all ChimeriVax ^tMvirus is total.It comprises (i) 3' pole far-end trunk-ring structure successively from 3' end, assuming that rise strand RNA synthetic promoter function and be conservative in all flaviviruss, (ii) two conserved sequence elements CS1 and CS2, the nucleotide sequence homology of height is shared with all mosquito matchmaker flaviviruss, and (iii) is for West Africa yellow fever virus strain, three copy tumor-necrosis factor glycoproteins element (RS) (Chambersetal. that comprise YF17D vaccine virus uniqueness, that be positioned at 3 ' UTR upstream portion, Annu.Rev.Microbiol.44:649-688,1990).3 ' UTR also comprises the trunk-ring structure of many predictions, the structure in the non-conservative district of such as RS member downstream, as shown in Figure 1B.

The normally short attenuation of 3 ' UTR sudden change of the present invention is deleted, such as, be less than 30 Nucleotide (such as 1,2,3 etc., until 29 (such as 2-25,3-20,4-15,5-10 or 6-8 Nucleotide length)).When being introduced separately into vaccine candidate strain or combining with other Attenuating mutations, new mutant of the present invention can cause the attenuation of extra level, thus provides the method for fine setting vaccine candidate strain attenuation.In some embodiments, the short deletion of 3 ' UTR of the present invention is designed so that the secondary structure of one or more prediction trunk structure in 3 ' UTR and/or the one-piece construction instability of 3'UTR.Except deletion, the sudden change in described structure also can comprise the similar replacement causing trunk structure instability.In certain embodiments, the non-conservative district that the trunk-ring structure carrying out sudden change of the present invention is positioned at 3 ' UTR maybe can tolerate the conserved regions (such as CS2) of described sudden change.Such as, with regard to yellow fever virus (such as YF17D) 3 ' UTR, no matter be complete yellow fever virus or the chimeric background based on yellow fever virus, the sudden change of trunk unstability can be present in any one or more trunk structures shown in Figure 1B, Figure 1B shows four these type of examples (dA, dB, dC and dD) deleted, and hereafter has further description.Like this, except the example that these are concrete, in yellow fever virus, other example of 3 ' UTR sudden change comprises and comprises shown following 1-2 of such as Figure 1B, 3-8 is individual, 4-7 is individual or the sudden change of the backbone sequences of 5-6 Nucleotide (from 5 ' to 3 ' direction is read): TGGAG, CTCCA, GACAG, TTGTC, AGTTT, GGCTG, CAGCC, AACCTGG, TTCTGGG, CTACCACC, GGTGGTAG, GGGGTCT, AGACCCT, AGTGG and TTGACG.

Except the sudden change of trunk unstability, sudden change of the present invention also comprises other the short deletion in 3 ' UTR.Such as, the present invention includes some sudden change fallen in Δ 30 mutational range, as mentioned above.Thus, such as, the present invention includes length in this region is 1,2,3 etc., until 29 (such as 1-25,2-20,3-15,4-14,5-13,6-12,7-11,8-10 or 9) any sudden change of surviving of Nucleotide.As concrete example, the present invention includes and delete d7, this region wherein in YF17D deletes following Nucleotide: 345-351 position Nucleotide (AAGACGG; Number from first Nucleotide of 3'UTR, after the UGA terminator codon of virus O RF; Figure 1A).The sudden change comprising from then on sequence 3 ' or 5 ' end deletion such as 1,2,3,4 or 5 other Nucleotide is also included within the present invention.In other embodiments, the short deletion in conserved sequence CS1 and CS2 is included in the present invention.These sudden changes can comprise such as delete these sequences 1-29,2-25,3-20,4-15,5-10 or 6-8 Nucleotide.As two concrete examples (hereafter having further description), from the CS2 of the special 3'UTR of YF17D, delete 360-364 position Nucleotide (GGTTA; CS2d5; Figure 1A) and/or 360-375 position Nucleotide (GGTTAGAGGAGACCCT; CS2d16; Figure 1A).The sudden change comprising from then on sequence 3 ' or 5 ' end deletion such as 1,2,3,4 or 5 other Nucleotide is also included within the present invention.For other flavivirus 3 ' UTR, similar sudden change can be built based on the secondary structure of 3 ' UTR.Deliver the prediction of the secondary structure about other flavivirus 3'UTR, such as (consult such as Proutskietal. about dengue fever virus, KUN and TBE virus, VirusRes.64:107-123,1999) and HCV (consult such as Kolykhalovetal., J.Virol.70:3363-3371,1996).In addition, the numerous 3'UTR nucleotide sequences representing many jaundice strains of whole four kinds of primary serum complex bodys (YF, JE, singapore hemorrhagic fever and TBE) can obtain from GenBank.The sequence of other virus strain measures by Virus Sequencing.Available standards software (such as mfold or RNAfold program) is easy to the secondary structure of these sequences of prediction to disclose the potential trunk-ring structure that can carry out mutagenesis.

As hereafter discussed further, 3 ' UTR sudden change of the present invention can be used for for attenuated vaccine candidate provides further attenuation.Therefore, one or more 3 ' UTR as herein described can be built suddenly change (such as d7, dB, dC and/or dD) thus provide higher level of security for YF17D yellow fever virus vaccine strain.Optional, 3 ' UTR sudden change can be included in this virus strain together with one or more other Attenuating mutations, (any one or more of such as 48-61,127-131 and 196-283 position coating amino acid in such as virus envelope protein hinge area, the such as replacement of the 279th amino acids, or in yellow fever virus with the 204th of 1 type dengue fever virus the, 252,253,257, the replacement (consulting such as WO03/103571) of one or more coating amino acids corresponding to 258 and 261 amino acids; The hinge area of flavivirus envelope albumen is between structural domain I and II; Consult such as Reyetal., Nature375:291-298,1995) amino acid (the film spiral part of such as membranin, in yellow fever virus membranin, such as with west Nile virus membranin the 66th corresponding amino acid) or the Attenuating mutations of any capsid protein described herein or envelope protein mutation (aminoacid replacement of such as corresponding with west Nile virus the 138th, 176,177,244,264,280,313,316,380 and 440 amino acids position, independent or combination).

In fact, likely by sudden change special in 3 ' UTR or delete with (as mentioned below) in capsid gene, in prM gene (the such as chimeric M66 of YF-Japanese encephalitis chimeric M5 or M60 or YF-WN or surrounding amino acid place (see PCT/US2005/037369 and hereafter)) or E protein in known make the site of flavivirus attenuation one or more suddenly change or delete and combine.E gene comprises Functional domains, and amino acid change wherein can affect function and thus reduce toxicity, as HurrelbrinkandMcMinn, Adv.VirusDis.60:1-42, described in 2003.The E protein functional zone that can produce suitable attenuated vaccine together with can deleting/suddenly change with 3 ' UTR described in the application after insertion mutation comprise: receptor binding domain a) Domain III outside surface estimated, b) the molecular hinge district between structural domain I and II, which determines the sour dependency conformational change of E protein in endosome (endosome) and reduces viral internalization efficiency; C) interface of prM/M albumen and E protein, is exposed to after resetting from dimer to tripolymer after low ph value in endosome and forms the region with the interface of prM/M E protein; D) top of the Fusion domain of domain II, it relates to the fusion with endosome film in internalization active procedure; And e) trunk-anchor district, it also functionally relates to the conformational change of E protein in sour induced fusion active procedure.

3 ' UTR sudden change also can be included in chimeric flavirirus vaccines Candidate Strain.(hereafter further describe) in one embodiment, one or more 3 ' UTR as herein described suddenlys change the capsid protein and the membranin of non-structural protein bletilla west Nile virus (NY99) and the vaccine candidate strain of envelope protein that are included in containing yellow fever virus (YF17D) (referred to here as ChimeriVax ^tM-WN02) in, its envelope protein has comprised the Attenuating mutations (replacement of the 107th, 316 and 440; WO2004/045529; Also can consult hereafter).Thus, the present invention includes the ChimeriVax also comprising such as d7, dB, dC, dD or any other 3 ' UTR sudden change (optional, with one or more capsid or other coating mutation combination, as described herein) as herein described ^tM-WN-02.In other embodiments, membranin and envelope protein from another kind of flavivirus, such as japanese encephalitis virus (such as SA14-14-2), dengue fever virus (1 type, 2 types, 3 types or 4 type dengue fever viruss) or other flavivirus described herein.

As yellow fever virus vaccine mentioned above, 3 ' UTR sudden change can optionally be included in chimeric flavirirus together with other Attenuating mutations one or more, (such as with yellow fever virus 48-61 in such as virus envelope protein hinge area, any one or more amino acid places that 127-131 and 196-283 amino acids is corresponding, such as the 279th amino acids, or with 1 type dengue fever virus the 204th in envelope protein, 252, 253, 257, the one or more amino acid whose replacement (consulting such as WO03/103571) that 258 and 261 amino acids are corresponding), amino acid (the film spiral part of such as membranin in membranin, such as with west Nile virus membranin the 66th corresponding amino acid), or any capsid protein described herein or envelope protein mutation are (such as with west Nile virus the 138th, 176, 177, 244, the aminoacid replacement of the position that 264 and 280 amino acids are corresponding, independent or combination) Attenuating mutations.The object lesson comprising the highly attenuated vaccine of dissimilar Attenuating mutations combination is documented in hereinafter.These C2 represented in wherein capsid protein delete to delete with d7, dB or the dD in 3'UTR and combine, or change the Chimeric Yellow Fever-west Nile virus variant of E#5 combinatorial association of (E176, E177 and E280 amino acid change) with the amino acid in envelope protein.

envelope mutations

Just as further discussed below, we to find in capsid protein short deletes sudden change and also can be used for for the vaccine candidate strain of attenuation provides further attenuation.Thus, the present invention includes in capsid protein the flavivirus (comprise chimeric flavirirus, such as comprised the vaccine candidate strain of other Attenuating mutations) comprising short deletion (such as 1,2,3 or 4 amino acid whose deletions).The example of this type of sudden change, with regard to YF17D viral capsid proteins, comprises the survived deletion (see Fig. 2 A) affecting protein spiral I.The object lesson of this type of sudden change is sudden change C2, and it comprises deletes amino acid PSR(Fig. 2 A from spiral I).Can test viability and the attenuation of other short sudden change in this region, they are also included within the present invention.The capsid protein sequence of other flavivirus is delivered, such as, about TBE is viral, WN is viral, KUN, JE are viral and dengue fever virus (such as Pletnevetal., Virology174:250-263,1990).

As mentioned above 3 ' UTR sudden change, can capsid protein sudden change be introduced in yellow fever virus vaccine strain (such as YF17D), optional and any one or more following mutation combination: 3 ' UTR as described herein suddenlys change (such as d7, dB, dC, dD or its variant), Attenuating mutations (such as 48-61 in virus envelope protein hinge area, any one or more of 127-131 and 196-283 position coating amino acid, such as the 279th amino acids, or with the 204th of 1 type dengue fever virus the in yellow fever virus, 252, 253, 257, the one or more amino acid whose replacement (consulting such as WO03/103571) that 258 and 261 amino acids are corresponding), amino acid (the film spiral part of such as membranin in yellow fever virus membranin, such as with west Nile virus membranin the 66th corresponding amino acid), or any envelope protein mutation described herein is (such as with west Nile virus the 138th, 176, 177, 244, the aminoacid replacement of 264 and 280 corresponding positions, independent or combination) Attenuating mutations.

Similar, capsid protein sudden change can be introduced in chimeric flavirirus.(hereafter have further record) in one embodiment, one or more envelope mutations as herein described is included in vaccine candidate strain (referred to here as ChimeriVax ^tM-WN02) in, described vaccine candidate strain comprises the capsid protein of yellow fever virus (YF17D) and the membranin of non-structural protein bletilla west Nile virus (NY99) and envelope protein, and its envelope protein has comprised the Attenuating mutations (replacement of the 107th, 316 and 440; WO2004/045529; Also consult hereafter).Thus, the present invention includes and also comprise such as C2 sudden change (optional and one or more 3 ' UTR and/or other coating mutation combinations, as described herein, such as C2 deletes the structure deleting with d7, dB or dD in 3'UTR or combine with the E#5 that amino acid in envelope protein changes and has characterized combination) ChimeriVax ^tM-WN-02.In other embodiments, membranin and envelope protein from another kind of flavivirus, such as japanese encephalitis virus (such as SA14-14-2), dengue fever virus (1 type, 2 types, 3 types or 4 type dengue fever viruss) or other flavivirus described herein.In other embodiments, as mentioned above, natural (non-chimeric) flavivirus is introduced in this type of sudden change.

In addition, 3 ' UTR sudden change can optionally be included in the chimeric flavirirus with a place or other Attenuating mutations of many places, Attenuating mutations in such as virus envelope protein hinge area is (such as with yellow fever virus 48-61, 127-131 and 196-283 position coating amino acid such as the 279th amino acids corresponding any one or more amino acid whose replacements, or with 1 type dengue fever virus the 204th in envelope protein, 252, 253, 257, the one or more amino acid whose replacement (consulting such as WO03/103571) that 258 and 261 amino acids are corresponding), amino acid whose Attenuating mutations (the film spiral part of such as membranin in membranin, such as with west Nile virus membranin the 66th corresponding amino acid), or any envelope protein mutation described herein is (such as with west Nile virus the 138th, 176, 177, 244, 264, 280, 313, 316, the aminoacid replacement of the position that 380 and 440 amino acids are corresponding, independent or combination).

coating suddenlys change

As described elsewhere herein, the existing record of some coating sudden change is said for flavivirus, and such as yellow fever virus (such as YF17D) and chimeric flavirirus are attenuations.These comprise hinge region mutations (such as with yellow fever virus 48-61,127-131,170,173,200,299,305,380 and replacement (the Hurrelbrinketal. of the replacement of position corresponding to 196-283 amino acids and the residue of domain II hydrophobic pocket lining; Adv.VirusRes.60:1-42,2003; Modisetal., Proc.Natl.Acad.Sci.U.S.A.100:6986-6991,2003; See Fig. 3), comprise the 52nd and 200 residues, the 279th amino acids (based in the chimeric background of yellow fever virus) of japanese encephalitis virus and the 204th, 252,253,257,258 and 261 amino acids (consulting such as WO03/103571) of 1 type dengue fever virus in yellow fever virus situation), and the replacement (consulting such as WO2004/045529) of west Nile virus envelope protein the 107th, 316 and 440 amino acids.

As hereafter discussed further, in an embodiment, we find that sudden change in flavivirus envelope sequence (such as replacing) also can provide the means of the attenuation of finely tuning attenuated flavivirus.Therefore, the present invention includes the flavivirus (such as yellow fever virus or chimeric flavirirus, as described herein) comprising and can be used for one or more envelope protein mutation of finely tuning candidate vaccine attenuation.The example of this type of sudden change comprises the replacement of the position corresponding with west Nile virus the 138th, 176,177,244,264 and 280 amino acids and the combination (such as the 176th, 177 and 280 of these sudden changes; 176th, 177,244,264 and 280; With the 138th, 176,177 and 280).As described herein, such as these make the attenuation of attenuated vaccine candidate have a little coating sudden change declined can be included in the equivalent site of yellow fever virus vaccine (such as based on the vaccine of YF17D) or chimeric flavirirus.Optional, be included together with these sudden changes can suddenly change with other coating one or more as herein described, capsid, film and/or 3 ' UTR.

The all sudden changes recorded about capsid gene and 3'UTR all can suddenly change with these coatings (with affecting a coating residue of attenuation or multiple residue according to the show) combine to produce the virus with att phenotype, namely its viscerotropism tropism compares low (namely the bringing out lower viremia in host) of often kind of parental virus do not combined.Such as, C2 mutant (table 2) can with E#7(table 4) or the combination such as dC, dD.3'UTR delete (table 1) can with C2 or E#7 virus combination.

the sudden change that can be included together with envelope protein mutation with 3 ' UTR of the present invention, capsid protein

Except the above-mentioned Attenuating mutations in a place or many places, flavivirus of the present invention can comprise other Attenuating mutations.Although to be referred in suddenling change with three classes included by the present invention the combination of each above, this part describes in more detail these sudden changes.

The example of sudden change that the flavivirus (comprising chimeric flavirirus) comprising the present invention's sudden change can comprise comprises sudden change in envelope protein hinge area or the sudden change of some membranin.Specifically, found that some envelope protein hinge region mutations reduces viscerotropism tropism.The polypeptide chain of envelope protein is folded into three unique structural domains: central domain (structural domain I), dimerization domain (domain II) and immunoglobulin-like modular structural domains (Domain III).Hinge area is between structural domain I and II, in virus after receptor-mediated endocytosis is taken in, when being exposed to acid pH, through conformational change (therefore called after " hinge "), cause the trimerical formation of envelope protein, it relates to the fusion of virus and endosome film.Before conformational change, protein exists with dimeric form.

Many coating amino acids are present in hinge area, comprise 48-61,127-131 and 196-283 amino acids (Hurrelbrinketal., Adv.VirusRes.60:1-42,2003 of such as yellow fever virus; Reyetal., Nature375:291-298,1995).The Attenuating mutations of the amino acid (and the corresponding amino acid in other flavivirus envelope albumen) of these amino acid any or immediate vicinity all can be present in virus of the present invention.Interested is especially amino acid in the hydrophobic pocket of hinge area (Modis etc. deliver before printing on the net on May 20th, 2003,10.1073/pnas.0832193100.PNAS|June10,2003|vol.100|no.12|6986-6991).As concrete example, the 204th the amino acid whose replacement of envelope protein (becoming R from K in 1 type dengue fever virus) in the chimeric flavirirus hinge area hydrophobic pocket comprising the 1 type dengue fever virus sequence inserting yellow fever virus vector causes attenuation.This replaces the structural modification causing envelope protein, makes a coating monomer and the intermolecular hydrogen bonding between another in wild-type protein be destroyed and replace with intramolecular interaction new in monomer.Thus, can replace with other intramolecular interaction increased in hydrophobic pocket, cause attenuation.The replacement of E202K, E204K, E252V, E253L, E257E, E258G and E261H can be comprised with mutation combination of the present invention, this type of example suddenling change/replace of building in hydrophobic pocket.

Except the sudden change of above-mentioned 3 ' UTR, capsid and/or coating, flavivirus of the present invention also can comprise a place in membranin or many places Attenuating mutations, such as, the amino acid place corresponding with west Nile virus membranin the 66th amino acids and/or other amino acid place in the film spiral predicted (such as at any one or more amino acid places corresponding with west Nile virus 40-75 amino acids).As concrete example, when west Nile virus membranin, the 66th amino acids (being leucine in wild-type west Nile virus) of membranin can use another kind of amino acid, and such as proline(Pro) replaces.Except proline(Pro), other hydrophobic amino acid can be used, such as Isoleucine, methionine(Met) or α-amino-isovaleric acid, or p1 amino acid, such as L-Ala or glycine replace membranin the 66th wild-type amino acid.As other example, west Nile virus the 60th, 61,62,63 and/or 64 (or the corresponding position in other flavivirus) amino acid can replace, independent or combination mutually, with the 66th mutation combination and/or with other mutation combination.The example of the replacement of these positions comprises: the 60th arginine becomes glycine, the 61st α-amino-isovaleric acid becomes L-Ala, the 62nd α-amino-isovaleric acid becomes L-glutamic acid or methionine(Met), the 63rd phenylalanine becomes Serine and the 64th α-amino-isovaleric acid becomes Isoleucine.Another example comprises ChimeriVax ^tMthe 60th of the membranin that in-JE virus, JE is special is become the sudden change of halfcystine by arginine, find that this sudden change improves the genetic stability of vaccine during extensive preparation.The extracellular domain that we also provide M albumen first has the evidence of critical function meaning, because ChimeriVax ^tMthe change that the M5 residue place glutamine of-JE becomes proline(Pro) improves the pH threshold value (consulting application PCT/US2005/037369) of infection.

Except an above-mentioned place or the sudden change of many places membranin, virus of the present invention also can comprise a place or other sudden change of many places.Such as, when west Nile virus, other sudden change described can in the region of west Nile virus envelope protein the 107th (such as L becomes F), the 316th (such as A becomes V) or the 440th (such as K becomes R) (or its combination).Thus, described sudden change can in the 102-112 position of such as west Nile virus envelope protein, the 138th (such as E becomes K), the 176th (such as Y becomes V), the 177th (such as T becomes A), the 244th (such as E becomes G), the 264th (such as Q becomes H), the 280th (such as K becomes M), 311-321 position and/or 435-445 position one or more amino acid places.As concrete example, by the sequence (GenBank numbering AF196835) of west Nile virus strain NY99-flamingo382-99 as reference, 107th Methionin can replace with phenylalanine, 316th L-Ala can replace with α-amino-isovaleric acid, and/or the 440th Methionin can replace with arginine.Corresponding sudden change can be built equally in other flavivirus.

In addition, virus of the present invention can also comprise may attenuation or may not attenuation but in other side other sudden change any for vaccine useful (such as vaccine preparation), such as, naturally can accumulate in virus propagation process and be the amino acid change in Nucleotide change in desirable UTR or structural protein or Nonstructural Protein.Such as, at ChimeriVax during we observe under serum-free condition extensive preparation process recently ^tMin-JE vaccine, the 60th residue R of accumulation becomes the amino acid change of C.This change does not affect immunogenicity or attenuation, but it is become the accumulation of the undesirable reverse of wildtype residues in envelope protein (E107) by its growth velocity of raising and prevention and made viral steady.

Standard method can be used in virus of the present invention, to build sudden change, such as site-directed mutagenesis.Above-described sudden change has deletion and replaces, but can use the sudden change of other type in the present invention equally, such as inserts.In addition, as mentioned above, sudden change can Individual existence or be present in background of other sudden change of a place or many places.In addition, except above-mentioned concrete amino acid, can also replace with other amino acid, such as can cause the amino acid of above-mentioned amino acid whose conservative change.Conservative replacement typically comprises the replacement in following group: glycine, L-Ala, α-amino-isovaleric acid, Isoleucine and leucine; Aspartic acid, L-glutamic acid, l-asparagine and glutamine; Serine and Threonine; Methionin and arginine; And phenylalanine and tyrosine.In addition, conservative all can select with nonconservative two kinds of changes, and the E protein x-ray structure caused based on they of computer forecast (using protein structure modeling software) changes the attenuating effects analyzing them.

Virus of the present invention (comprising mosaic) can use this area standard method to prepare.Such as, the RNA molecule corresponding with viral genome can be introduced primary cell, chicken embryo or diploid cell line, then can therefrom (or from it clear liquid in) purifying progeny virus.The another kind of method that can be used for producing virus uses heteroploid cell, such as Vero cell (Yasumuraetal., NihonRinsho21:1201-1215,1963).In the method, the nucleic acid molecule (such as RNA molecule) corresponding with viral genome is introduced heteroploid cell, results virus from cell cultured nutrient solution, with nuclease (endonuclease of both such as degradation of dna and RNA, such as Benzonase ^tM; U.S. Patent No. 5,173,418) virus of process results, by the viral concentration (such as by be that the filter of such as 500kDa carries out ultrafiltration with molecular weight cut-off) through nuclease process, then prepares concentrated virus, for inoculating purposes.The details of the method are shown in WO03/060088A2, in this income as a reference.

Virus of the present invention can be used as the crowd that primary prevention agent (primaryprophylacticagent) puts on infection risk, or can be used as secondary preparation (secondaryagent) and be used for the treatment of the patient be infected.Because described virus is attenuation, they are particularly suitable for being applied to " risky individuality ", such as the elderly, children or HIV person.Described vaccine also can be used for veterinary field, such as, carry out the inoculation for the vaccine inoculation of West Nile Virus infection or birds (such as valuable, the in imminent danger or birds raised and train, are respectively such as flamingo, bald eagle and goose) to horse.In addition, vaccine of the present invention can containing the virus comprising specific sudden change, and such as embedded virus, is mixed with the virus lacking described sudden change.

The preparation of virus of the present invention can use this area standard method to carry out.Many pharmacy prepared for vaccine can accept solution to be well-known and to be easy to (consult such as Remington ' sPharmaceuticalSciences (the 18th edition) for the present invention after those skilled in the art's amendment, editor A.Gennaro, 1990, MackPublishingCo., Easton, PA).In two concrete examples, virus is prepared in the minimum essential medium EarleShi salt (MEME) containing 7.5% lactose and 2.5% human serum albumin or containing in the MEME of 10% sorbyl alcohol., virus can simply can accept to dilute in solution at physiology, such as Sterile Saline or sterile buffered saline.In another example, virus can such as carry out using and preparing in the mode identical with yellow jack 17D Vaccine, such as, as the clarified suspension through infecting chicken embryo tissue or the liquid from the cell culture results infecting embedded virus.

Vaccine of the present invention can use method well-known in the art to use, and vaccine appropriate amount to be administered can be easy to determine by those skilled in the art.Determine that what is that virus appropriate amount to be administered is such as following because usually determining by considering, the stature size of the experimenter of such as virus to be administered and holistic health.Such as, virus of the present invention can be mixed with aseptic aqueous solution, at volume be 0.1 to 1.0ml potion in containing 10 ²to 10 ⁸individual, such as 10 ³to 10 ⁷individual infectious unit (such as plaque forming unit or TCID), is used by such as intramuscular, subcutaneous or intracutaneous path.In addition, because flavivirus may through mucous membrane path, such as oral routes infects human host (Gresikova etc., " Tick-borneEncephalitis ", at the TheArboviruses that Monath compiles, in EcologyandEpidemiology, CRCPress, BocaRaton, Florida, 1988, VolumeIV, 177-203), so virus is also used by mucous membrane path.In addition, vaccine of the present invention can single dose be used, or optional, uses to comprise to use amount of initiator (primingdose), after such as 2-6 month, use booster dose (boosterdose) subsequently, suitably determined by those skilled in the art.

Optional, the adjuvant that those skilled in the art will know that can be used when using virus of the present invention.Can be used for the immunogenic adjuvant of enhanced virus comprise such as liposome formulation agent, synthetic adjuvant such as (such as QS21), Muramyl dipeptide, monophosphoryl lipid A (monophosphoryllipidA), poly-phosphine piperazine, CpG ODN or seem by activating cells on the surface or intracellular nucleic film other molecule of working of Toll-like receptor (TLR) molecule on the surface.Although these adjuvants are typically for strengthening the immunne response to inactivated vaccine, they also can use together with living vaccine.Both the agonist of TLR or antagonist all can be used for the situation of living vaccine.In virus through mucous membrane path, such as, when oral route is sent, the mutant derivative of mucosal adjuvants such as E.coli LT (LT) or LT can be used as adjuvant.In addition, the gene that coding can be had the cytokine of adjuvanticity inserts virus.Like this, the gene of encode cytokines such as GM-CSF, IL-2, IL-12, IL-13 or IL-5 can be inserted to produce the vaccine causing immunne response to strengthen together with foreign antigen genes, or immunity moderation is oriented to the response of cell, body fluid or mucous membrane with more specific.

At dengue fever virus and/or when comprising the chimeric flavirirus of the membranin of dengue fever virus and envelope protein, the best for them inoculates the immunogenic induction that can comprise for whole four kinds of Dengue serotypes, and virus of the present invention can be used for preparing tetravalent vaccine.As described herein, the place or many places sudden change that reduce viscerotropism tropism can be comprised for the arbitrary of this type of tetravalence preparaton or all virus.Described virus in any time point mixing of process for preparation to form tetravalence preparation, or can sequentially can be used.When tetravalent vaccine, often kind of virus of equal parts can be used.Or, use the amount of the often kind of different virus existed in vaccine can different (WO03/101397A2).

Flavivirus of the present invention also can be used for delivery of heterologous genes product, and such as vaccine antigen or other therapeutical agent (consult such as WO02/102828; U.S. Patent No. 6,589,531; And WO02/072835).

Part of the present invention is based on following experimental result.

Experimental result and embodiment

Background and general introduction

In one embodiment of the invention, ChimeriVax is referred to herein as ^tMall sudden changes described above are built in the chimeric flavirirus vaccines Candidate Strain of-WN02, described chimeric flavirirus vaccines Candidate Strain comprises the capsid of yellow fever virus (YF17D) and the cephacoria of non-structural protein bletilla west Nile virus (NY99) and envelope protein, hereafter has further record.This vaccine candidate strain is before clinical and the test of I phase clinical study.Although it shows mouse and rhesus monkey (rhesusmonkey) camber attenuation and has immunogenicity, in some human volunteers (N=45) that it tests in macaque (cynomolgusmonkey) with in the I phase according to judgement inoculate afterwards viremia compared with contrast yellow jack (YF) 17D Vaccine, induce more active copying.Even if its well-tolerated and have high degree of immunogenicity in the I phase tests, according to viremia, we are undertaken studying to improve ChimeriVax further by the means of specific mutagenesis ^tMthe security feature of-WN02, object reaches and ChimeriVax ^tMthe viremia that-WN02 variant is compared in meiofauna (hamster) has slight reduction.Have employed three kinds of mutafacient system, be discussed in respectively in following examples.In first method, segment Nucleotide is deleted the 3 '-non-translational region (3 ' UTR) introducing virus.In the second approach, amino acid deletion is introduced capsid protein.In the third method, specific attenuating amino acid is replaced the envelope protein introducing virus.As hereafter and herein other is locally discussed further, the sudden change of these types can combination with one another (and with other attenuation or the beneficial mutation of other side combine) build virus of the present invention.

ChimeriVax ^TM-WN02

Initial YF17D/WN mosaic comprises the wild-type prM-E gene order from WN virus N Y99 strain, called after ChimeriVax ^tM-WN01 is use standard double-mass model system constructing (Arroyoetal., J.Virol.78:12497-12507,2004).Plasmid pYF5 ' 3 ' IV/SA14-14-2(is comprised for ChimeriVax ^tM5 ' and 3 ' part of the cDNA of-JE vaccine virus) and pYFM5.2/SA14-14-2(comprise ChimeriVax ^tMthe large section middle portion of-JEcDNA) in the corresponding cDNA sequence of JE specificity prM-E gene order NY99WN parent replace.ChimeriVax ^tM-WN01 virus is following to be obtained, and by the fragment of Ligation in vitro from gained pYWN5'3'N1 Δ 3 and pYWN5.2/5 plasmid, obtains full length DNA template, subsequently in-vitro transcription with gained rna transcription thing transfected Vero cells.New embedded virus demonstrates and compares mouse and the remarkable attenuation of rhesus monkey with WNNY99 and YF17D, although it preserves the spared nerve virulence to adult mice.By the table 3 that sees below at residue E107, E316 and E440(of coating (E) albumen) place introduces the change of three SA14-14-2JE vaccine specific acidic amino acids, makes its further attenuation.Comprise two novel plasmid called after pYWN5'3'NF3 Δs 2 and the pYWN5.2316/440#2 of these sudden changes.Based on called after ChimeriVax ^tMthe test result of gained three mutant in mouse and monkey of-WN02, reach a conclusion, it obtains abundant attenuation, thus can test further in I clinical trial phase.Described test is carried out (for 10 in normal adults ⁵the dosage of inoculation of pfu, N=30; And for 10 ³the dosage of pfu, N=15).Unexpectedly, in the inoculation volunteer of two dosage groups, there is the sick toxicaemia of several human hair, be significantly higher than YF-Vax contrast (until high 3.5 times) statistically.This illustrates, although described vaccine well-tolerated and have immunity, still can develop to obtain the ChimeriVax of attenuation more further ^tM-WN vaccine variant.High viremia may indicate the over-replicate of virus in peripheral organ, and may presentation portion divide inoculator that the risk of bleeding occurs, similar to the yellow jack of classics, or owing to there is the danger of encephalitis through hemato encephalic barrier, can play a driving role to this based on to the high viremia of the understanding causing encephalitis fiavivirus.

Thus, ChimeriVax ^tMthe unique suboptimum parameter of-WN02 vaccine is slightly higher than viremia after the expection inoculation observed in a part of human volunteer, and this may indicate the over-replicate (viscerotropism tropism) in peripheral organ.Because initial ChimeriVax ^tM-WN02 virus has been highly attenuated vaccine candidate strain, and the wild-type strain isolated of non-toxicity power, so we attempt to identify the new mutant that can not make the excessive attenuation of vaccine.We find in suitable animal model (we used hamster in hereinafter described experiment) and in people, stop high viremia to occur subsequently but significantly do not reduce the sudden change of effect.

New ChimeriVax ^tMthe generation of-WN04 Candidate Strain and the experimental procedure involved by sign comprise:

-build the plasmid DNA of suddenling change containing expectation.Specifically, initial ChimeriVax is introduced in all sudden changes as follows ^tM-WN02 plasmid pYWN5'3'NF3 Δ 2 and pYWN5.2316/440#2, the mutagenesis that the oligonucleotide utilizing simple or overlapping round pcr to carry out standard instructs, connects into these plasmids by gained PCR primer subsequently and selects mutant plasmid by clone in intestinal bacteria and to individual clones order-checking.

The EagI-BspEI large fragment of pYWN5'3' and the pYWN5.2 series plasmids that-Ligation in vitro is suitable, to obtain full-length cDNA template, uses XhoI linearizing subsequently.

-carry out full length DNA template with SP6RNA polysaccharase in-vitro transcription with produce synthesis, have infective RNA.

-by producing saltant type ChimeriVax with fat lipofectamine (lipofectamine) or electroporation transfection Vero cell ^tM-WN04 virus also gathers in the crops the first-generation (P1) Virus Sample, carries out later passages subsequently to obtain P2 virus stocks in serum free medium.

-viability of mutant virus is confirmed by monitoring cytopathic effect (CPE), the plaque assay of cell conditioned medium liquid and the detection of sensitive T-PCR to viral RNA.

-confirm by the total order-checking of the horizontal virus genome RNA of P2 the existence (and carrying out initial analysis genetic stability by carrying out order-checking to the virus being passaged to P5 level) expecting sudden change.

-analyze plaque growth and morphology characteristic by the standardized titration of P2 virus in Vero cell.

-analyze inoculation 10 by viremia after the inoculation of measuring 1-9 days ⁵viscerotropism tropism in the Syria hamster of pfu/ agent P2 viral sample; By the 30th day with standard 50% plaque reduce in and test (PRNT ₅₀) serum titer of measuring WN virus-specific neutralizing antibody analyzes immunogenicity.

As mentioned below, we are at ChimeriVax ^tMintroduce deletion in the 3'UTR of-WN02 virus or capsid (C) albumen, attempt to reach very slight attenuating effects in the highly attenuated vaccine candidate strain verified at this.The segment grown by 5-16 in 3'UTR Nucleotide is deleted or in PROTEIN C, 3 amino acid whose deletions reach this object.It is short (5-6 the Nucleotide) deletion (Proutskietal., J.Gen.Virol.78:1543-1549,1999) design based on the Secondary structure predietion of YF17D virus 3 ' UTR that some in 3'UTR are deleted.Object is that specific some certain computers prediction trunk-ring structure making to be positioned in the middle part of 3'UTR, outside any conserved sequence elements is unstable.In the third method, we are at ChimeriVax ^tMintroduce other SA14-14-2 Attenuating mutations in the E protein of-WN02, this and we are for from ChimeriVax ^tM-WN01 develops ChimeriVax ^tMthe method of-WN02 is identical.The effect that these are modified has been monitored in Hamster model.Qualification draws ChimeriVax ^tM-WN04 variant causes viscerotropism tropism to have weakening a little of expectation in hamster.

Structure ChimeriVax is deleted by introducing specificity in 3'UTR ^tM-WN04-3'UTR Candidate Strain

As mentioned above, Figure 1A shows all ChimeriVax ^tMvirus the formation of YF17D specificity 3'UTR shared.It comprises 3 '-pole far-end trunk-ring structure that (i) guards for all flaviviruss successively from 3 ' end, (ii) two conserved sequence elements CS1 and CS2, and (iii) is positioned at three copy tumor-necrosis factor glycoproteins element (RS) (Chambersetal. of 3 ' UTR upstream portion, Annu.Rev.Microbiol.44:649-688,1990).A valuable feature of the deletion in this region is their stability, because the spontaneous in virus replication is in fact impossible.We are at ChimeriVax ^tMintroduce in-WN02 virus (use plasmid pYWN5'3'NF3 Δ 2), produce new ChimeriVax ^tM11 places of-WN04-3'UTR Candidate Strain delete as shown in Figure 1A, comprising:

The dRS of-removal three RS elements deletes (the 18-164 position Nucleotide ATAACCGGG...-...TCCACAC of YF17D3'UTR; First 3'UTR Nucleotide open numbering after the TGA terminator codon of virus O RF);

-everywhere 5-6 Nucleotide segment delete: be present in the dA(229-234 position Nucleotide between RS and CS2, GCAGTG), dB(256-260 position Nucleotide, CAGGT), dC(293-297 position Nucleotide, and dD(308-312 position Nucleotide CCAGA), CGGAG), be designed for and make the particular computer prediction trunk-ring shown in Fig. 2 B/puppet knot (pseudoknot) structural instability (Proutskietal., J.Gen.Virol.78:1543-1549,1999);

The large section of-105 Nucleotide is deleted, d105(218-321 position Nucleotide, TAAGCT...-...CCGCTA), it removes most of Nucleotide between RS and CS2 (similar deletion obtain wild-type DEN4 tolerance but significantly reduce in vitro and in vivo copy (Menetal., J.Virol.70:3930-3937,1996), therefore we estimate that it is for ChimeriVax ^tM-WN02 can be excessive attenuation);

The deletion of-30 Nucleotide, d30(322-351 position Nucleotide, CCACCC...-...GACGG); be positioned at the upstream of CS2; imitating initial record makes the Δ 30 of wild-type DEN4 virus attenuation suddenly change (Menetal., J.Virol.70:3930-3937,1996; U.S. Patent No. 6,184,024B1);

The deletion that-two places are less, d7(345-351 position Nucleotide, AAGACGG) and d14(338-351 position Nucleotide, TGGTAGAAAGACGG), be positioned at Δ 30 district, this being carried out to test is that sudden change can make ChimeriVax in vitro and/or in vivo because we estimate above-mentioned d30 ^tMthe excessive attenuation of-WN02;

The segment of two 5 and 16 Nucleotide in place in-CS2 district is deleted, called after CS2d5(360-364 position Nucleotide, GGTTA) and CS2d16(360-375 position Nucleotide, GGTTAGAGGAGACCCT).

By monitoring the viability of mutant virus to the careful microscopic examination of P1 and P2 period in generation CPE, and be passaged to P5 subsequently to assess can survive mutant genetic stability and determine whether any second point mutation can recover the viability of the apparent mutant that can not live.These observationss obtain the confirmation of the intracellular plaque assay of RT-PCR and Vero.Plaque assay also creates about mutant virus titre (the intracellular growth characteristics of its instruction Vero) and the important information about plaque metamorphosis (it is caused by the deletion introduced).In P2 and P5 level, virus genomic relevant portion of can surviving is checked order.The results are summarized in table 1 of these experiments.

According to the data in table 1, we reach a conclusion:

1) although it is that large section is deleted that dRS deletes, it does not cause significant attenuation in vitro, because mutant virus produces greatly and clearly plaque, with ChimeriVax ^tM-WN02LP contrast (large plaque contrast; Consult the footnote 1 of table 1) plaque similar, and grow to 6x10 ⁶the relatively high titre of PFU/ml.This result is expected, because similar deletion does not almost affect (Bredenbeeketal., J.Gen.Virol.84:1261-1268,2003) YF17D virus replication in vitro, and similar large section deletion does not reduce ChimeriVax ^tMthe neurovirulence of-JE virus in suckling mouse.

2) according to the judgement of plaque form, segment is deleted dA, dB, dC and dD and is caused significant attenuation in vitro.The plaque of whole four kinds of mutant virus reduce the size of; The plaque of dD virus is opaque (plaque of LP and SPWN02 two kinds contrast is all transparent).Virus grew to more than 10 in P2 generation ⁶the relatively high titre of PFU/ml, this is desirable feature (table 1 for preparation; Except the titre of dA mutant, it can not count so can not determine because the plaque formed in this experiment is too little).It is important that these results show that the trunk-ring/pseudoknot structure (Figure 1B) predicted really exists and copies for flavivirus.The wild-type DEN4 virus (Menetal., J.Virol.70:3930-3937,1996) of deleting with the large section in tolerance this region, CS2 upstream is contrary, contains segment and deletes the large section d105 deletion of all structural elements of institute's target for ChimeriVax ^tM-WN02 virus is excessive attenuation (lethal).

3) be in close proximity to CS2 before region in suddenly change (Menetal., J.Virol.70:3930-3937,1996 with Δ 30; U.S. Patent No. 6,184,024B1) similar d30 deletes and shorter deletion d14 is lethal.ChimeriVax is obtained in this region ^tMunique sudden change of-WN02 tolerance is that segment d7 deletes.It has attenuation, because it morphologically makes plaque reduce.

4) segment CS2d5 and CS2d16 deletes and has medium attenuating effects in vitro, because the mutant comprising described deletion forms the opaque plaque of medium-sized size.Estimate that these sudden change impacts comprise the 10th, the prediction trunk-ring structure of 708 Nucleotide place XbaI restriction site sequences, as shown in Figure 1B.

In these experiments, the segment that we demonstrate outside 3'UTR Conserved Elements first deletes the attenuation that can provide to a certain degree.In addition, we demonstrate the specific item forecast trunk-ring/pseudoknot structure instability that makes first and cause attenuation.In addition, in the 3'UTR of flavivirus comprising YF17D virus, there is the trunk-ring/pseudoknot structure of many predictions, for the attenuating effects obtaining a sequence provides diversified chance.Region between these structures or structure can be used as the target location that segment that those skilled in the art can introduce is deleted at present.According to our discovery, can estimate that the mutagenesis of these structures any all can provide attenuation to a certain degree at present.From a sequence effect, carry out selecting the mutant can chosen and have and expect attenuation degree.

Table 1.ChimeriVax ^tM-WN04-3'UTR deletes the external feature of mutant

Sudden change	The viability of mutant	P2 titre, PFU/ml	Plaque form
				dRS	Can existence	6x10 6	Large and transparent
dA	Can existence	N/D 2	Small
				dB	Can existence	6.3x10 6	Less than SP contrast
dC	Can existence	1.5x10 6	Small
				dD	Can existence	6.6x10 6	Medium-sized, opaque
CS2d5	Can existence	1.2x10 7	Medium-sized, opaque
				CS2d16	Can existence	5.3x10 6	Medium-sized, opaque
d7	Can existence	5.2x10 6	Less than SP contrast
				d14	Can not survive	N/A	Without plaque
d30	Can not survive	N/A	Without plaque
				d105	Can not survive	N/A	Without plaque
WN02LP contrasts 1			Large and transparent
				WN02SP contrasts 1			Little and transparent

1LP with SP contrast virus (only for comparing plaque form in plaque assay) is separated the ChimeriVax from preparation by plaque purification in advance ^tM-WN02P5 viral sample, described sample is large plaque (LP) group and little plaque (SP) group; SP variant seems the accumulation becoming the change of this monoamino-acid of Pro owing to M66Leu.

2N/D – after infection the 5th day in order to when accurate metering carries out neutral red staining because plaque is too little undetermined.

Should it is mentioned that the real secondary structure comprising 3 ' UTR of the flavivirus of YF17D virus be unknown, because they are in the intact virus background not have effective means experimentally to confirm, therefore the prediction delivered, such as Proutski and colleague thereof, about the prediction (Figure 1B) of YF17D, may be incorrect.Can predict and form many selectable structure (Zukeretal. in relatively long RNA molecule, N.A.R.19:2707-2714,2001), and possible be that different structure (in normal chain or minus strand) is formed in the different steps in vial life cycle and works.Real structure can be subject to various false knot and form (Olsthoornetal., RNA7:1370-1377,2001) and remote RNA interact (such as RNA cyclisation and other interact (Alvarezetal., J.Virol.79:6631-6643,2005)) and possible with host with the interactional impact of RNA of virus protein.Further complicated explanation is carried out in order to predict the outcome to the theoretical computer delivered, frequent use manual operations, such as local sequence initial fold and subsequently initial predict is put in the structure of longer RNA sequence, artificially N is used in initial fold step, and the preferred structural element of subjective selection (such as Mutebietal., J.Virol.78:9652-9665,2004).For this reason, we use conventional Zuker prediction algorithm to carry out 3 ' the UTRRNA sequence of folding YF17D.As shown in Figure 1 C, the Proutsky prediction shown in it from Figure 1B is different for the optimum structure of prediction.Segment importantly in Figure 1A and 1B deletes natural YF17D the best (Fig. 1 C) and the suboptimum structural instability that dA, dB, dC, dD, d7 and d14 make prediction usually.Fig. 1 D shows an example (for dC mutant) of described altered optimum structure.On the contrary, CS2d5 and CS2d16 deletes (Figure 1A and 1B) and does not significantly change best natural structure, show that these deletions can by changing the sequence of CS2 own but not 3 ' UTR structure or by changing some suboptimum structure, making virus attenuation (for ChimeriVax ^tMthe attenuation of-WN is confirmed in Hamster model).Therefore, even if some deletes based on Proutski structure prediction design (Figure 1B), their real effect may owing to making different structure element but not prediction trunk-ring in Figure 1B is unstable.

In order to analyze genetic stability, dC mutant is passaged to P5 level from P2 in serum-free Vero cell and to P5 Virus Sequencing after, find the spontaneous length (277-300 position Nucleotide, TCTGGGACCTCCCACCCCA is deleted) increasing 24 Nucleotide of deletion of 5 Nucleotide.(other deletes (dRS, d7, CS2d5, CS2d16, dA, dB and dD) is stable in same genetic stability goes down to posterity.) effect that increases of the size of deleting is that the secondary structure of prediction becomes and initial best YF17D structural similitude (Fig. 1 E; Compare with Fig. 1 C).This spontaneous change looks like a kind of adaptive change of cell cultures.P2 and P5 two kinds of variants are all highly attenuated and have immunogenic (footnote 4 see table 5) in hamster.Therefore, the P5 variant of dC mutant may have desirable vaccine phenotype.By introducing the ChimeriVax that specific deletion is carried out in the capsid protein C that YF17D is special ^tMthe structure of-WN04-C Candidate Strain

The Computer Analysis of the YF17DC protein structure that we use ProteinPredict with Protean method to carry out dopes its general formation not to be had substantive different from (Fig. 2 B) of TBE.We infer ChimeriVax ^tMlarge section in-WN02 internal protein is deleted and will most possibly be caused excessive attenuation.Therefore, 3-4 amino acid whose 5 place's segments that we introduce as shown in Figure 2 B delete (residue of deletion indicates with square frame).The ChimeriVax introducing and contained complete C protein gene will be deleted ^tM-WN02 plasmid pYWN5'3 ' NF3 Δ 2.(C1-3 are deleted at three places; Each length 3 amino acid) be positioned at Kofleretal., J.Virol.76:3534-3543, about the identical general areas that TBE records in 2002.But, we determine the position of sudden change to test the importance of certain structural features: C1 deletes the upstream being positioned at both spiral I of central hydrophibic section and prediction, and C2 only affects spiral I, and C3 expectation can disturb both spiral I and central hydrophibic section.In addition, C4 deletes, and (long 4 amino acid) are designed to the spiral III of the prediction of target C-terminal, the part of protein belt positive charge, and C5 deletion (3 amino acid) (it further eliminates the NS2b/NS3-viral protein cleavage sites of the C-terminal being positioned at born of the same parents' formal protein between spiral III and IV; Introducing it is the polyprotein manufacturing deficiency whether sudden change in order to determine any second site can compensate expectation).

The vitro characterization of WN04-C mutant the results are summarized in table 2.Only have the existence of C1 and C2 sudden change physical efficiency, and C3-C5 deletion is lethal.Although the strong deleterious effects of C5 sudden change is not astonishing, what is interesting is C-terminal, it is lethal that the segment of the part of protein belt positive charge deletes (C4), shows that the sequence/structural modification in this region is that virus institute is insupportable.It is also lethal for the most surprisingly observing C3 deletion, because it is arranged in the identical general areas that TBE virus background tolerates the deletion of large section.The existence of deleting in C1 and C2 variant is confirmed by order-checking.In P2 and P5 level virus, the sequencing result of complete structure protein region is as shown in table 2.Although C1 variant is in residue M14 and E313 place cumulative change, (they show as heterogeneity in P5 generation, are not then (think that the change at E313 place is previous at ChimeriVax in P2 generation ^tMthe adaptation of the serum-free viral growth condition of accumulation is observed in-WN02)), but C2 variant seems it is stable in heredity.Rear a kind of virus all comprises the heterogeneity at residue M71 place at P2 and P5, but in succeeding generations, the ratio (~ 80%) of mutant and non-mutant does not change.According to the judgement of plaque form, C1 variant and ChimeriVax ^tM-WN02 compares does not have attenuation, and C2 variant looks like attenuation, because which forms little plaque, indicates the importance of spiral I.Two kinds of mutant all grow paramount titre (about 10 in Vero cell ⁷pFU/ml).Do not have the data of prior disclosure to show such segment deletion can make flavivirus attenuation or there is practical value.The success of viable C1 and C2 mutant is produced as in our study proves YF17D virus or ChimeriVax ^tMthe segment deletion that vaccine virus can tolerate upstream, central hydrophibic district and alpha-helix I section start provides evidence.

Table 2.ChimeriVax ^tMthe vitro characterization of-WN04-C mutant

1WN02 contrast virus (only in plaque assay for comparing plaque form) uses ChimeriVax ^tMthe P1 sample that-WN02 external rna transcription thing transfectional cell obtains.

2 check order to confirm that expection is deleted and checks the existence of other amino acid any change/heterogeneity by consistent method to complete structure protein region (C-prM-E gene).

By introducing the ChimeriVax that the special change of other SA14-14-2 is carried out in coating (E) albumen ^tMthe structure of-WN04-E Candidate Strain

In wild-type JE virus (Nakayama) and SA14-14-2 vaccine strain, in different E protein residues and WNNY99, the residue of corresponding position is as shown in table 3.As mentioned above, ChimeriVax ^tMthe expected degree attenuation of-WN02 vaccine variant is reach by special for three SA14-14-2 residue E107, E316 and E440 being introduced in YF17D/WN mosaic that originally built, that comprise the special prM-E gene of wild-type NY99 strain (WN01 virus) at first.ChimeriVax ^tM-WN02(and WN01) also comprise E227 serine residue consistent in SA14-14-2 with NY99 two-strain.

Table 3. is for reducing ChimeriVax ^tMthe viscerotropism tropism of-WN021 will at ChimeriVax ^tMthe residue of the SA14-14-2JE vaccine specific in the envelope protein E of combination in-WN04

1 is present in ChimeriVax ^tMthe special residue of 1SA-14-14-2 in-WN02 indicates with runic; Note, residue E227 is identical (Serine) in SA14-14-2 and WNNY99, does not therefore need to be changed by special mutagenesis.

2 amino acid number are according to the numbering of WN virus E protein.

Previously at all plasmid (Arroyoetal. selecting to build in WN02 vaccine candidate strain process, J.Virol.78:12497-12507,2004) and our those plasmids (two basic pYWN5'3' and pYWN5.2 constructs) of building recently as shown in Figure 4.In embedded virus E protein, the expectation set of SA14-14-2 sudden change is by obtaining the specific paired DNA fragmentation from suitable pYWN5'3' and pYWN5.2 plasmid via EagI restriction site Ligation in vitro in E gene, such as ChimeriVax ^tM-WN02 is connected generation by pYWN5'3'NF3 Δ 2 with pYWN5.2316/440#2.Some combination was previously tested in mouse and monkey, have selected WN02 Candidate Strain thus for carrying out testing (Arroyoetal., J.Virol.78:12497-12507,2004) in people further.M66 sudden change (the 66th residue leucine of M albumen becomes proline(Pro)) that we mix two new pYWN5'3' plasmids is ChimeriVax in extensive preparation process ^tMthe cell cultures adaptive change of accumulation in-WN02 vaccine.Which reduce Virus plaque size, but the neurovirulence of mouse is not affected (Arroyoetal., J.Virol.78:12497-12507,2004).According to us recently from the result that I phase human trial and other monkey are tested, that reduce the viral viscerotropism tropism to primate and the sudden change looked like thus vaccine beneficial properties, improve security.Because current ChimeriVax ^tM-WN02 vaccine is the mixture of large plaque and little plaque, causes the viremia of a little higher than expection in some people, so carried out overwork to reduce viscerotropism tropism.Such as, recently plaque purification has been carried out to little plaque variant, just tested in monkey at present.The new structure variant with the mutation combination previously do not tested is described below.

The ChimeriVax that table 4. is new ^tM-WN04-E variant: the sudden change of introducing and the vitro characterization of virus

1 all virus is all obtained by transfected Vero cells; In Virus Name, " A " represents the variation in full length DNA Template preparation (3 fragments connect), and " R " represents the virus repeating transfection and obtain; The virus of drawing top shadow is selected to be used for testing further in hamster.

The residue that 2WN02SA14-14-2 is special: E107,227,316 and 440; It is the known cell cultures adaptive change reducing WN02 Virus plaque size that M66 changes.

3L+S, the apparent mixture (in 1R virus) of large plaque and little plaque; S, little plaque; <L+S, plaque looks like the mixture of large plaque and little plaque, but size of population is slightly smaller than 1R virus (L+S).

4 check order to confirm that the sudden change expected (confirms, except as otherwise noted) one by one by consistent method to complete structure protein region (C-prM-E gene); List other amino acid change/heterogeneity detected.

Relevant new ChimeriVax ^tMthe viability of-WN04-E construct and the Data Summary of vitro characterization are in table 4.Virus 11, wherein we attempt combining the special residue of all 10 SA14-14-2, it seems it is inviable, because it does not induce CPE after infection, in plaque assay, do not form plaque in later passages process, and its geneome RNA does not detect in cell conditioned medium liquid by sensitive RT-PCR reaction.Other new virus comprising 5 to 9 SA14-14-2 changes listed in table 4 is viable (3,4,5,6 and No. 7 all samples).Great majority wherein it seems slight attenuation, because their plaque is slightly less than the plaque of WN02 contrast (viral 1R), although they grow beyond 10 ⁶-10 ⁷the relatively high titre of pfu/ml; Unique exception is virus-4, and owing to adding E138 and M66 sudden change simultaneously, to make it it seems be excessive attenuation (small plaque and very low titre) for it.

Confirm that the expection SA14-14-2 in virus 3,5 and 7 changes by consistent order-checking.Virus 6 is strange, because its 6R sample lacks one of expection sudden change when P2 checks order: it has wild type glutamic acid and replaces glycine (it also lacks two the silent nucleotide changes specially introduced in E244 triplet downstream to form SphI restriction site) at residue E244 place.Another sample, 6A, has α-amino-isovaleric acid at residue E244 place, is different from wild-type WN and SA14-14-2 sequence (it has the SphI site of expection).These changes in virus sequence are unexpected, because for generation of the confirmation of the special district of the whole virus in the pYWN5.2/8mut plasmid preparation of 6R, 6A and 11 external rna transcription things through order-checking.Change that likely E244SA14-14-2 is special (independent or combine some other change such as E264 time) for ChimeriVax ^tM-WN is lethal, and this is soluble, and why virus 11 can not be recovered.The modification of this residue recovering to detect in 6R and the 6A sample of viability is attributable to unstable in bacterium of the mispairing (most likely the situation of viral 6A) of varial polymerases in virus replication, pYWN5.2/8mut plasmid or plasmid and checks order the light contamination of another bacterial clone do not disclosed.The P2 virus checked order is relatively homogeneous, and except the accumulation that some virus starts to demonstrate some other sudden changes, wherein some are that expected (by the change of G to R, it is ChimeriVax to such as E313 ^tMthe known serum-free cell culture adaptive change not affecting biological phenotype of-WN).More different sudden change is have accumulated in the process being passaged to P5 level.According to these observationss, the virus of P2 level 3,5, draw dash area in 6A and 7(table 4) select to be used in hamster, carry out relevant viscerotropism tropism/immunogenic further test.

ChimeriVax ^tMthe viscerotropism tropism that-WN04 variant carries out in hamster and Analysis of Immunogenicity

ChimeriVax is proved as sensitive meiofauna model with mouse ^tMthe decline (this is the important indicator of attenuation) of-WN vaccine candidate strain neurovirulence and its high immunogenicity (Arroyoetal., J.Virol.78:12497-12507,2004).But, this model can not be used for the viscerotropism tropism level (this is another important symbol of attenuation) predicted in monkey and the mankind, because mosaic does not bring out detectable viremia in mouse.Some flavivirus brings out high-caliber viremia in hamster, as nearest about (Teshetal., Emerg.Inf.Dis.8:1392-1397,2002) shown by wild-type WN virus.We use ChimeriVax recently ^tM-WN02 vaccine (mixture of large plaque and little plaque virus) and demonstrate that these viruses cause in female Syrian hamsters through the research that LP and the SP variant of plaque purification carries out have good dependency between viremia and the viremia observed in human volunteer and macaque.Specifically, the LP variant less for people's attenuation induces the viremia that can easily detect in hamster, and attenuation more SP virus then induces viremia that is very low or that can't detect.We utilize this new meiofauna model whether to reduce viscerotropism tropism to study above-mentioned WN04 sudden change, and do not hinder the generation of effective anti-WN immunne response.

All codes are all carry out according to the requirement of NIH regarding assay room animal humanity process under the scheme of AcambisIACUC approval.10 are inoculated to surrounding female Syrian hamsters in age subcutaneous (SC) ⁵the selected ChimeriVax of pfu ^tM-WN04 Candidate Strain and WN02LP and SP contrast virus or about 10 ⁴the YF17D of pfu, is measured the viremia (animal take a blood sample and measures institute with plaque assay and gather in the crops virus titer in serum under narcosis) of the 1st, 3,5,7 and 9 day subsequently and is neutralized by the minimizing of standard 50% plaque and test (PRNT in individual animal ₅₀) the measurement antibody response of the 30th day.Result is as shown in table 5.

Table 5. female Syrian hamsters SC inoculation after to ChimeriVax ^tMthe viremia of-WN04 variant and antibody response

1 inoculation volume: 100 μ l; Dosage of inoculation: ChimeriVax ^tM-WN04 virus and WN02LP and SP contrast are 10 ⁵pfu, and YF17D (YF-VAX) is about 10 ⁴pfu; Simulation inoculation animal accepts 100 μ l diluted medium (MEM, 50%FBS).

2 detection levels: 25PFU/ml.

3 passed through PRNT at the 30th day ₅₀measure the NAT for following virus: inoculation ChimeriVax ^tMall groups of-WN variant is ChimeriVax ^tM-WN02 or YF-VAX group are YF17D, or simulation inoculation group is above two-strain.

4 test the 5th generation (P5) sample of dC mutant in the hamster experiment separated, and wherein delete spontaneously to be increased to 24 Nucleotide (seeing above).Virus keeps highly attenuated (viremia average peak is 25pfu/ml, and average duration is 3.5 days) and has the immunogenicity (PRNT of the 33rd day ₅₀gMT is 452).CS2d5 and CS2d16P2 virus is also tested, has found the PRNT of similar highly attenuated and immunogenicity (viremia average peak (pfu/ml)/viremia average duration (my god)/the 33rd day ₅₀gMT is respectively 137/2.75/127 and 343/2.25/905).When immunity carries out attack test with wild-type WN in about 10 months afterwards, contrast contrary with the little plaque of WN02, comprise the immune all animals crossed of dCP5 with these WN04 variants and be obtained for firm protection.

ChimeriVax ^tM-WN02LP contrast (the vaccine variant of attenuation deficiency) induces the peak value viremia from 1,650 to 2,925PFU/ml scope (average out to 2,285PFU/ml) in the hamster of 5 inoculations.These animals had 5,120 to 10 at the 30th day, the highest WN NAT of 240 scopes (GMT6,760).WN02SP to merely hit the viremia of not bringing out and can detect to impinge upon 5 animals 4; 1 hamster is the detection limits 25PFU/ml of this assay method in the low-level viremia that the 3rd day detects.The response of WN specific antibody is very low in these animals.It can't detect (titre <10) in 2 hamsters; Other 3 the low antibody titerss with 40-320.What is interesting is, inoculation YF-VAX does not cause the viremia that can detect but creates high YF specificity Neutralizing antibody response (320-10,240PRNT ₅₀titre; GMT2,150).As is expected, simulation inoculation animal did not have WN or YF specificity neutralizing antibody at the 30th day.

Have small reduction in order to reach to the viscerotropism tropism of primate, we identify a set of ChimeriVax at desirable hope in Hamster model ^tM-WN04 variant, the viremia scope that they cause can low than caused by LP virus, but bring out the immunne response higher than SP virus simultaneously.At the ChimeriVax of 11 kinds of tests ^tMin-WN04 virus, according to the judgement of viremia after inoculation, it seems that great majority to have attenuation (table 5) to a certain degree (average virus mass formed by blood stasis titre peak value at <25 within the scope of Isosorbide-5-Nitrae 60pfu/ml) in vivo at least compared with LP virus.In the variant that attenuation is less, there is C protein to delete mutant C1,3'UTR delete mutant dRS and WN04-E variant #3 and #5.These are viral-induced height Neutralizing antibody response (GMT1,110-2,560).A kind of virus, WN04-E variant #6A, proves significant excess attenuation according to very low viremia and weak both antibody responses.Undoubtedly, rear a kind of virus can be got rid of outside the scope of carrying out further testing in monkey/mankind.There is the ChimeriVax of advantageous particularly characteristic ^tM-WN04 Candidate Strain is that capsid protein deletes mutant C2,3'UTR segment deletion mutant d7, dB, dC and dD and E#7 variant.These viruses cause viremia medium to violent minimizing (average virus mass formed by blood stasis titre peak value is in <25 to 680PFU/ml scope) in hamster, but do not hinder strong immunne response (neutralizing antibody GMT370-2,940).

In order to prove Vaccine effectiveness, within the 62nd day after immunity, inject 4x10 in all animal peritoneal ⁵pFU has the wild-type WN virus of virulence (NY382/99 strain) to carry out attack test.According to not there is attacking rear viremia (measuring in the serum of the 1st, 3,5,7 and 9 day results), lose weight, the judgement of symptom or death; there are all animals of high titre WN neutralizing antibody (at the 30th day); (see table 5) particularly in WN04-C1, C2, dRS, d7, dB, dC, dD, E#3, E#5, E#7 and WN02LP control group, is obtained for and protects completely.At least some animal, particularly E#6A of other group, WN02SP contrast, YF-VAX and simulation group, they do not have height WN NAT, according to the judgement of at least one above-mentioned parameter, are not protected.All there is high viremia at 1-5 days (viremia titre peak value is up to 9.75x10 in all YF-VAX and simulation immune animal ⁵pfu/ml).These animals got sick of great majority, lose weight, and have 2 animal deads in YF-VAX group.2 animals of E#6 group and 1 animal of WN02SP group demonstrated low-level viremia at 1-2 days.ChimeriVax ^tMthe impact that sudden change in-WN04 variant grows in liver cell virus

To suddenly change other evidence that the viscerotropism tropism that causes reduces to obtain WN04, we analyze some most promising ChimeriVax ^tM-WN04 variant (according to the data selection presented above, such as, viremia in hamster is low and immunogenicity is high; In table 5) growth kinetics in human liver cell oncocyte system HepG2.Because YF virus is close liver property (hepatotropic) virus, we wish to see WN04 variant and ChimeriVax ^tM-WN02LP virus (vaccine of attenuation deficiency) compares the minimizing copied.Infect HepG2 cell monolayer with the MOI of 0.005PFU/ml, that gathers in the crops aliquots containig every day contains vial supernatant (to the 10th day), and the plaque assay then by carrying out in Vero cell measures virus titer.This attenuation WN04 variant included by experiment has 3'UTR to delete mutant d7, dB, dC and dD, and capsid protein deletes mutant C2(and the less mutant C1 of attenuation as other contrast), and envelope protein mutation body E#7.Except C1 mutant, the duplicating efficiency of other WN04 virus all is not as good as WN02LP virus (Fig. 5), but the growth fraction WN02SP variant (thinking that it is the vaccine variant for the excessive attenuation of the mankind) of great majority (except dD) in them is good.This shows that some WN04 sudden change can reduce the close liver tropism of vaccine in human body, and this is very desirable feature.This experiment show further the benefit showing E#7, d7, dC and dD Candidate Strain the most significantly copying minimizing compared with WN02LP, and wherein dD mutant may be that close liver tropism is minimum.

ChimeriVax ^tM-WN04 double mutant; Become internal organ tropism and the Analysis of Immunogenicity that carry out in hamster

The combination of dissimilar Attenuating mutations should cause other attenuation and more reliable vaccine phenotype, and more impossible recovery is pathogenic.For this reason, four kinds of double mutant ChimeriVax are constructed ^tM-WN04 variant, wherein deletes C2 deletion with d7, dB or dD3'UTR and combines or combine with the E#5 mutation combination (change that other E176,177 and 280 SA14-14-2 are special) in envelope protein.DNA profiling for in-vitro transcription is with previously for the 5'3' of the single mutation construction of WN04 and the suitable part of 5.2 plasmids are connected acquisition by standard two fragment or three fragments.With these DNA profilings of SP6RNA polymerase transcription, and reclaim virus after with rna transcription thing electroporation Vero cell.Titration P2 virus in Vero cell.All four kinds of double mutants all show as because it produces small plaque (be less than WN02LP and SP contrast) in cell cultures and obtain strong attenuation.C2+E5 mutant has 1.3x10 ⁷the high titre of pfu/ml, and other 3 kinds of viruses (C2+d7, C2+dB and C2+dD) have 4.2-6.1x10 ⁵the middle titre (table 6) of pfu/ml.

Table 6.ChimeriVax ^tMthe vitro characterization of-WN04 double mutant

Virus

P2 titre, PFU/ml

The plaque form of the 5th day

C2+E5P2	1.3x10 7	Small (on average about 0.5mm)
			C2+d7P2	4.2x10 5	Small
C2+dB P2	6.1x10 5	Small
			C2+dD P2	6.1x10 5	Small
WN02LP contrasts	9x10 6	Greatly/little, transparent
			WN02SP contrasts	5.8x10 7	Little, transparent

In order to assess attenuation and immunogenicity, inoculate 10 to surrounding Syria hamster in age subcutaneous (SC) ⁵the double mutant of pfu, and WN02LP and SP contrast virus, or thinner (camouflage product).Measure viremia at 1-10 days, pass through PRNT at the 35th day ₅₀measure antibody response.Result as shown in Table 7.Four kinds of double mutants have replication, cause the low-level viremia that can detect in most animals, except C2+d7 only detects except viremia in an animal.These viruses are significant more attenuation (average peak viremia about 7,000pfu/ml) compared with WN02LP virus, and it seems more attenuation (such as comparing those numerical value in average peak viremia titre and table 5) compared with corresponding single mutant.Although be not all viremia detected in each animal, all hamsters all create high-caliber Neutralizing antibody response.The PRNT of C2+E5, C2+d7, C2+dB and C2+dD ₅₀gMT numerical value is 1:640,1:840,1:1 respectively, 280 and 1:640.

Table 7. female Syrian hamsters SC inoculation after for ChimeriVax ^tMthe viremia of-WN04 double mutant variant and antibody response

1 viremia limit of detection 50pfu/ml; Within 8-10 days, also tested, viremia do not detected.2 because not all animal all reaches the serum dilution that generation 50% neutralizes, so this numerical value may be significantly higher than shown.

In order to confirm provide protection, at the 36th day with 2x10 ⁵pfu/ agent is viral to the wild-type WN that injection height in animal peritoneal is lethal, and NY385/99 strain (more pathogenic than the NY382/99 used in experiment above) is to carry out attack test.Obtain protect completely with the animal of WN04 double mutant and WN02LP contrast immunity, because viremia (measuring at the 2nd, 4 and 6 day) and losing weight after there is not the attack of instruction disease.On the contrary, WN02SP and the immune control animal of camouflage product are then protected.They create viremia (peak response titers of WN02SP and camouflage product immune animal is respectively 250-3,000 and >2,000pfu/ml), show the symptom of disease and lose weight.1 and 4 death are had respectively in 5 animals of WN02SP group and 5 animals of camouflage product group.2 animals of WN02SP group survival and 1 animal paralysis of camouflage product group survival.

So, by using the uniqueness of three kinds of different methods of flavivirus attenuation to modify, and introduce dissimilar Attenuating mutations alone or in combination, we successfully generate and previous ChimeriVax ^tM-WN02 vaccine is compared more attenuation but is not the multiple ChimeriVax of excessive attenuation ^tM-WN04 Candidate Strain.

Claims (6)

1. comprise the chimeric flavirirus of YF17D strain yellow fever virus, the membranin wherein in this yellow fever virus and the envelope protein membranin of NY99 strain west Nile virus and envelope protein are replaced, and wherein this chimeric flavirirus comprises following sudden change:

A L107F, A316V and K440R in () envelope protein replace;

The deletion (C2) of (b) capsid protein 40-42 amino acids; With

C () is selected from the sudden change of lower group:

The deletion of lower group is selected from: 256-260 in (i) this chimeric flavirirus 3 ' non-translational region (3 ' UTR)

The position deletion (dB) of Nucleotide, the deletion (dD) of 308-312 position Nucleotide or the deletion (d7) of 345-351 position Nucleotide, wherein nucleotides number starts from first Nucleotide of 3 ' UTR after viral open reading-frame (ORF) (ORF) UGA terminator codon, or

(ii) Y176V, T177A and K280M in envelope protein replace.

2. the chimeric flavirirus of claim 1, the west Nile virus envelope protein of wherein said chimeric flavirirus comprises the replacement in the 138th coating amino acid further.

3. containing the chimeric flavirirus of claim 1 or 2 and the medicinal compositions of pharmaceutical acceptable carrier or thinner.

4. the purposes of composition in the medicine for the preparation of the flaviviridae infections in prevention or treatment experimenter of claim 3.

5. the purposes of claim 4, wherein said flaviviridae infections is West Nile Virus infection.

6. comprise the genomic nucleic acid molecule of the chimeric flavirirus of claim 1 or 2.